Saudi Journal of Biological Sciences xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Saudi Journal of Biological Sciences

journal homepage: www.sciencedirect .com

Original article

A comparative toxic effect of Cedrus deodara oil on larval proteincontents and its behavioral effect on larvae of mealworm beetle(Tenebrio molitor) (Coleoptera: Tenebrionidae)

http://dx.doi.org/10.1016/j.sjbs.2017.06.0051319-562X/� 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

⇑ Corresponding author at: Department of Zoology, Kohat University of Scienceand Technology, Kohat, Pakistan.

E-mail addresses: ishtiaq11@gmail.com, ishtiaq@kust.edu.pk (S.I. Anjum).

Peer review under responsibility of King Saud University.

Production and hosting by Elsevier

Please cite this article in press as: Buneri, I.D., et al. A comparative toxic effect of Cedrus deodara oil on larval protein contents and its behavioral elarvae of mealworm beetle (Tenebrio molitor) (Coleoptera: Tenebrionidae)Cedrus deodara oil –>. Saudi Journal of Biological Sciences (2017), http:/org/10.1016/j.sjbs.2017.06.005

Islam Dad Buneri a, Masarrat Yousuf a, Mohammad Attaullah b, Saifullah Afridi c, Syed Ishtiaq Anjumd,⇑,Habibullah Rana e, Naveed Ahmad f, Muhammad Amin a, Muhammad Tahir g, Mohammad Javed Ansari h

a Toxicology Laboratory, Department of Zoology, University of Karachi, Karachi, PakistanbDepartment of Zoology, Shaheed BB University, Sheringal, Upper Dir, PakistancCentre for Advanced Drug Research, COMSAT Institute of Information Technology Abbotabad, Abbotabad 22060, PakistandDepartment of Zoology, Kohat University of Science and Technology, Kohat, PakistaneBRC, University of Karachi, Karachi, PakistanfCentre of Excellence in Marine Biology, University of Karachi, Karachi, Pakistang Faculty of Marine Science, Lasbela University of Agriculture, Water and Marine Sciences, Uthal, Balochistan, PakistanhBee Research Chair, Department of Plant Protection, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history:Received 27 April 2017Revised 1 June 2017Accepted 5 June 2017Available online xxxx

Keywords:TenebrioMealwormsCedrus deodaraImidaclopridCarbosulfanProtein levelToxicity

Cedrus deodara (deodar) is practically used, as insect repellent, in the northern areas of Pakistan but nodata available therefore this study was conducted to evaluate the effectiveness of deodar oil as an alter-nate of conventional insecticides against the larval pest stage of mealworm beetle (Tenebrio molitor), byfeeding method. The aim of the study was to investigate the effectiveness of deodar oil as an alternate ofconventional insecticides against the larval pest stage of mealworm beetle (Tenebrio molitor), by feedingmethod. All tested chemicals showed efficacy against the pests. The LC50 was determined by probit anal-ysis and was found to be 3.41, 0.086 and 0.023% of larvae treated with deodar oil, Carbosulfan andImidacloprid respectively The LC50 treated larvae were subjected to the evaluation of protein activity,qualitatively and quantitatively. The protein level in tested insects was enhanced when treated withImidacloprid, Carbosulfan and deodar oil. The electrophoretic profile of treated insects showed morebands in insects treated with Cedrus deodara oil. This electrophoretic profile appeared in 4, 5, 7 and 8bands for tested chemicals including control. Antifeedant activity was observed for C. deodara as larvaewere deterred to feed on the food found in the container.� 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is anopen access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Synthetic insecticides have been found effective against storedproduct pests but proved to be hazardous to men and domesticanimals. The over reliance and illegitimate use of synthetic pesti-cides especially insecticides since last four decades have led to a

wide spectrum of pest problems like pest resistance to chemicals,resurgence of pests, residues in food and soil and risks to humanand animal health. Entomologists of the world are in struggle tointroduce alternatives of synthetics pesticides. They started workon plant products which possess biologically active components.About 1800 plants species have been described to contain proper-ties against insects (Jacobson, 1982; Grainge et al., 1984). Differentcompounds have been prepared from various parts of plants whichaffect different types of insects (Naqvi et al., 1994). Many plantspossess activities against stored grain pests. In the present study,Deodar oil was used for the first time in Pakistan against Meal-worms to evaluate their toxic effects. Cedrus deodara belongs tothe family Pinaceae, commonly called Deodar. It is found abun-dantly in the northern areas of Khyber Pakhtunkhwa, Pakistanand also in the Mediterranean and in the Western Himalayas.Cedrus oils are extracted from Cedrus deodara plants. The oil is

ffect on/dx.doi.

2 I.D. Buneri et al. / Saudi Journal of Biological Sciences xxx (2017) xxx–xxx

given a local name as Ranzra in KPK. The residents of KPK use thisoil as insect repellent in the infected livestock. Different parts ofthe plant have been used as medicines in the area for a long time.The essential oil is distilled from the inner wood which is aromaticand is used on the feet of camels, cattle and horses as insect repel-lant (Gamble, 1922) and against houseflies and stored pests (Singhet al., 1984, 1989; Singh and Rao, 1985; Singh and Agarwal, 1988).Imidacloprid and Carbosulfan were used in this study as standardinsecticides. Mealworm was used as experimental insect in thestudy. Carbosulfan belongs to Carbamate family of insecticidesand is used to control insects, mites and nematodes. Carbosulfaninhibit cholinesterase activities in the target insects. The secondtested insecticide in the present study was Imidacloprid, a memberof Neonicotinoids insecticides, which belongs to compounds pre-pared from tobacco plants (Kim, 2006). Imidacloprid acts on nico-tinic acetylcholine receptor i.e. produce harmful effects on thenervous system of the insects (Liu et al., 2004). This action over-stimulate the nervous system and as a result of continue move-ments of muscles, paralysis and death may occur in the targetinsects (Bloomquist, 2009). Chaudhary et al. (2011) determinedthe toxicity of essential oil of Cedrus deodara against diamondbackmoth, Plutella xylostella L. but did not give its biochemical findings.In the present work, toxicity assessment and biochemical analysisof Cedrus deodara oil along with two chemicals was carried out inlarvae of stored grain pest, Tenebrio molitor.

Table 1Dose stimulus percentile Tenebrio molitor larvae treated with Insecticides.

Deodar dosestimulus

Carbosulfan dosestimulus

Imidacloprid dosestimulus

Insects killedpercent

0.123291 0.003010474 0.001167389 10.326156 0.008064255 0.002802075 50.54795 0.013638875 0.004469656 100.825727 0.020661675 0.006465 161.027203 0.025775398 0.007868843 201.304193 0.03282654 0.009755126 251.615995 0.040786918 0.011831108 302.379413 0.060354905 0.016759464 403.413957 0.086999728 0.02319406 504.89831 0.125407418 0.032099141 607.212339 0.185573051 0.045470332 708.936638 0.230574186 0.055146846 7511.34645 0.293650274 0.068366394 8014.11497 0.366328121 0.083211822 8421.27039 0.554954348 0.120359243 9035.73477 0.938580555 0.191987903 9594.53329 2.514206188 0.460826953 99LC50 ?

3.413957LC50 ?0.086999728

LC50 ?0.02319406

Table 2Protein bands and their intensity.

Bands Control Imidacloprid Carbosulfan Deodar

1 +++ +++ +++ +++2 +++ +++ ─ +3 ─ ─ ─ +4 ─ � + +5 ++ +++ + +++6 � � +++ +++7 � � + +8 + ++ ++ ++

2. Materials and methods

Adult mealworms (Tenebrio molitor) were collected from differ-ent regions of Keamari Town Karachi, Pakistan. Karachi Port Trust(KPT) storage areas and different stored grain Shops were visited.Hand picking method was applied during collection. The insectswere then brought to the insectary of the Department of Zoology,University of Karachi. Methodology of Morales-Ramos et al.(2011) was followed for the rearing of Tenebrio molitor. About1Kg of bran and 1 kg of potato was taken inside large, deep, openedmouth bowl. For maintaining moisture, some amount of water wassprayed onto the bran and potatoes were embedded in the centerof bran. Insects were reared at room temperature of about 25-30C�and about 70% humidity. Tenebrio molitor were released intoplastic bowls and were protected from the attacks of insectivoresand other enemies. The mouth of the bowl was closed with thehelp of black muslin cloth for maintaining darkness and rubberband. The beetles were kept undisturbed for about 15 days duringwhich egg laying and hatching process occurred. Fresh bran wasprovided to larvae and after 6–7 days, the larvae were subjectedto experimental analysis. A stock solution of 12% for Deodar oiland 1% solution each for Imidacloprid and Carbosulfan was pre-pared by using Charles’s formula. After preliminary trials, five dif-ferent concentrations from stock solution were prepared for eachchemical. About 2 mL of specific concentration each compoundwas mixed /10 gram of bran in a small plastic bowl and then 20 lar-vae were released into it. The mouth of each bowl was closed withcloth. Non-treated insects i.e. Control and check was also main-tained for environmental effects. Mortality was noted after 48 hof treatment. LC50 was calculated according to Finney (1971)method and Biostat software. The live 1 g of LC50 treated insectswere kept in freezer. When the larval activities were completelydiminished, then the larvae were crushed in 5 mL de ionized waterwith the aid of mortar pestle. The whole crushed sample was takenin a test tube and subjected to homogenizer for homogenization at1000 rpm for 10 min. The homogenates was then centrifuged for30 min at speed of 4000 rpm. The supernatants were used for esti-mation of total protein and electrophoresis while the lower layerwas discarded. For the estimation of total protein contents, Ecol-

Please cite this article in press as: Buneri, I.D., et al. A comparative toxic effectlarvae of mealworm beetle (Tenebrio molitor) (Coleoptera: Tenebrionidae)Cedruorg/10.1016/j.sjbs.2017.06.005

ine� Kit #123119966314 and spectrophotometer model 721-2000 were used. Proteins were separated by sodium dodecyl sul-phate (SDS) page gel electrophoresis according to the method ofLaemmli (1970). Samples were boiled for 3–5 min at 93 �C in SDSsample buffer containing and then processed on 10% SDS gel. Fromeach sample, 10 mL was loaded per slot of a pre cast 10% polyacry-lamide gel. For the determination of protein mass, recombinantproteins (Bio-Rad) markers were used as molecular weightstandards.

3. Results

Five different concentrations (0.75, 1.5, 3, 6 and 12%) of deodaroil were used and mortality was noted after preliminary trials as17, 33, 49, 64, and 87% respectively. LC50 of Cedrus deodara wasdetermined by Probit method and found to be 3.4% (Tables 1, 3and Fig. 1). Lowest mortality was 17% caused by 0.75% oil whilehighest mortality was 87% due to 12% oil. The LC50 of Carbosulfanwas 0.086% (Table 1, 5 and Fig. 2). Five concentrations i.e. 0.5, 0.25,0.125, 0.0625 and 0.03125% caused 92, 75, 60, 42 and 26 percentmortality respectively. Minimum and maximum mortality was26 and 92 percent respectively. The LC50 of Imidacloprid was0.023% (Table 1 and Fig. 3). Five concentrations i.e. 0.00781,0.0156, 0.0313, 0.0625 and 0.125% caused 22%, 44%, 61%, 74%and 96% mortality respectively. The lowest concentration(0.023%) caused 50% mortality which shows effectiveness of Imida-cloprid against Tenebrio molitor larvae Table 4. By comparing thetoxicity of three insecticides, the order of effectiveness was foundas Imidacloprid > Carbosulfan > Deodara. Total protein contentswere found to be 2.80, 2.68 and 4.14 mg/mL in larvae treated with

of Cedrus deodara oil on larval protein contents and its behavioral effect ons deodara oil –>. Saudi Journal of Biological Sciences (2017), http://dx.doi.

Table 3Toxicity assessment of deodar against Tenebrio molitor larvae.

% Concentration Mean mortality Median S.D. S.E. Range

12 87 85 5.700877 2.549587 91.99719104─82.002808966 64 65 4.1833 1.870886 67.66693572─60.333064283 49 50 4.1833 1.870886 52.66693572─45.333064281.5 33 35 2.738613 1.224782 35.40057293─30.599427070.75 17 15 2.738613 1.224782 19.40057293─14.59942707Control 1 0 2.236068 1.00003 2.960059587─0.960059587Check 2 0 2.738613 1.224782 4.400572926─0.400572926

Fig. 1. Larvae treated with deodar. Response (0–100): Number of insects diedstimulus (�1 to 14): Specific concentration of deodar extract.

Fig. 2. Larvae treated with Carbosulfan. Response (0–100): Number of insects died.Stimulus (0–0.5): Specific concentration of Carbosulfan insecticide.

Fig. 3. Larvae treated with Imidacloprid. Response (0–100): Number of insects died.Stimulus (0–0.14): Specific concentration of Imidacloprid insecticide.

I.D. Buneri et al. / Saudi Journal of Biological Sciences xxx (2017) xxx–xxx 3

Please cite this article in press as: Buneri, I.D., et al. A comparative toxic effectlarvae of mealworm beetle (Tenebrio molitor) (Coleoptera: Tenebrionidae)Cedruorg/10.1016/j.sjbs.2017.06.005

Carbosulfan, Imidacloprid and deodar oil respectively Fig. 4. Whilein control and check, it was found as 2.30 and 2.12 mg/mL respec-tively Fig. 4. The total protein amount in LC50 treated insects wasenhanced as compared to the control while it was slightlydecreased (7%) in the check sample (Fig. 3). Enhancement in theprotein contents by the three chemicals was in the order of Deo-dar > Carbosulfan > Imidacloprid. Repellency test showed that thediet of larvae treated with Deodar oil repelled the insects. The lar-vae moved to the side of the container and avoid feeding (Fig. 6).The insect sample was subjected to electrophoresis. A maximumof eight and minimum of four bands of protein were appeared dur-ing electrophoresis (Fig. 5). Protein bands 1, 5 and 8 were observedand were common in all test samples. Band 2 was noted in control,Imidacloprid and deodar while missed in Carbosulfan treated sam-ples. Band 3 was noted in deodar sample only and was absent inthe remaining samples. Band 4 was missed in control and Imida-cloprid samples while appeared in case of Carbosulfan and deodartreated samples. Likewise, bands 6–7 were disappeared in controland Imidacloprid while appeared in Carbosulfan and deodar trea-ted samples. Deodar showed a maximum of eight bands, Carbosul-fan five bands, Imidacloprid 3 bands and control showed 3 bands(Table 2). It was observed that total numbers of protein bands wereincreased when Tenebrio molitor larvae exposed to Carbosulfan,Imidacloprid and deodar oil (see Fig. 5 and Table 2).

4. Discussion

Mortality of mealworms (Tenebrio molitor) and impact on pro-tein contents were the two main studied parameters in the presentresearch work. The toxic effects of chemicals seem to have affectedthe protein contents in the treated samples. The more proteinbands in the treated insects may indicate that the individual pro-tein was broken into many abnormal fragments.

The essential oils of Cedrus deodara plant was used against sec-ond instars of the diamond back moth (Plutella xylostella L.) aseffective larvicides (LC50 = 425 lg/mL) (Chaudhary et al., 2011).In the present research crude oils of Cedrus deodarwas used againstlast larval instar of Tenebrio molitor and LC50 was found as 3.4%.It isknown that the affectability of various insects’ pests species couldbe vary for a similar substance. The different in LC50value may bedue to the difference in species and method of application.Makhaik et al. (2005)used essential oil of Cedrus deodara againstmosquito species, Aedes aegypti and Culex quinquefasciatus and cal-culated LC50 as 2.5% against Culex quinquefasciatus after one hour ofexposure. These results are comparable in their effectiveness inrepelling different insect’s species. In the present investigation,crude oil was used and the LC50 was found as 3.4%. The high LC50

in the present investigation may be due to the differences in cuticleof insect species as coleopterous species have hard and thick cuti-cle than the integument of mosquitoes, stage of development ofinsect, method of application of oil and duration of treatment.Tenebrio absorbed more tested chemical that resulted in slight highLC50 values. Cedrus deodara has been found effective against a vari-ety of insects (Raguraman and Singh, 1997;Rahuman et al., 2009;

of Cedrus deodara oil on larval protein contents and its behavioral effect ons deodara oil –>. Saudi Journal of Biological Sciences (2017), http://dx.doi.

Table 4Toxicity assessment of Imidacloprid against Tenebrio molitor larvae.

% Concentration Mean mortality Median S.D. S.E. Range

0.125 96 95 4.1833 1.870886 99.66694–92.333060.0625 74 75 4.1833 1.870886 77.66694–70.333060.03125 61 60 4.1833 1.870886 64.66694–57.333060.015625 44 45 4.1833 1.870886 47.66694–40.333060.007813 22 20 2.738613 1.224782 24.40057–19.59943Control 00 00 00 00 00–00

Table 5Toxicity assessment of Carbosulfan against Tenebrio molitor larvae.

% Concentration Mean mortality Median S.D. S.E. Range

0.5 92 90 2.738612788 1.224782105 94.40057293–89.599427070.25 75 75 3.535533906 1.581186899 78.09912632–71.900873680.125 60 60 3.535533906 1.581186899 63.09912632–56.900873680.0625 42 45 4.472135955 2.000060803 45.92011917–38.079880830.03125 26 25 4.183300133 1.870885569 29.66693572–22.33306428Control 00 00 00 00 00–00

Fig. 4. Shows total protein (mg/mL) in treated, Control and Check mealwormslarvae.

Fig. 5. Electrophoretic profile of Tenebrio molitor larvae treated with insecticides.10% SDS-PAGE (Left to right) represents, Lane 1(M): 6.5–200 kD Protein Marker;Lane 2–3: Control (duplicates), Lane 4–5 (A); Imidacloprid (duplicates). Lane 6–7(B): Cedrus deodara (duplicates); Lane 8–9(C): Carbosulfan (duplicates) respectively.Upper panel (Top to bottom) numbers represents the measureable protein bands intest samples (reduced): Lower panel in Lane 1(M): represent marker band mass inKD (kilo Dalton) respectively.

4 I.D. Buneri et al. / Saudi Journal of Biological Sciences xxx (2017) xxx–xxx

Rao and Singh, 2002;Delbeke et al., 1997). The findings suggestingthat formulation chemistry can investigate the longevity of repel-lent activity and should be developed into commercial repellentproducts.

Gregory et al. (1994) assessed the effects of bran treated withCarbaryl, Dimethoate and Chlorpyrifos against adult and larvalstage of Tenebrio molitor. According to them, Chlorpyrifos was mosteffective in terms of mortality of adult and larvae of Tenebriorespectively followed by Dimethoate and Carbaryl. By increasingthe amount of bran bait of Carbaryl and Dimethoate, the insectshowed increased response. After 1–5 days of treatment, they cal-culated ED50 for adults and larvae as 0.128 and 0.550 respectively.In the present investigation, Imidacloprid was foundmore effectivefollowed by Carbosulfan and deodar oil. Ezz and Fahmy (2009)used mineral oil i.e. Alboleum as well as an IGR, Admiral (pyriprox-yfen) on adult female mealybug Ferrisia virgata (Cockerel) anddetermined that total protein was enhanced by Admiral up to29.2%. The amount of protein enhanced in the present study was21.7% (2.80 mg/mL), 16% (2.68 mg/mL) and 80% (4.14 mg/mL) inlarvae treated with Carbosulfan, Imidacloprid and Deodar oilrespectively is similar to the previous study as in both, the amountof protein was increased. Marron et al. (2003) reported that facingstressful state like starvation, organisms develop two compen-satory mechanisms whether to store extra energy or by reducingtheir metabolic rate. This study is very close to the present demon-stration as protein contents enhanced in all treated insects so themechanism of the increased protein in treated group may be due

Please cite this article in press as: Buneri, I.D., et al. A comparative toxic effectlarvae of mealworm beetle (Tenebrio molitor) (Coleoptera: Tenebrionidae)Cedruorg/10.1016/j.sjbs.2017.06.005

to starvation, and it is evident from the behavior of the larvaewhich move to the side of the container with deodar containingbran. Such type of protein sparing strategy was usually presentin all insects during stress (McCue et al., 2015). Additionally, uponexposure of insects and other animals to various stressors, the pro-duction of most proteins declines, but heat shock proteins (HSPs)increase (King and MacRae, 2015; Chapuis et al., 2011). However,increased levels of protein oxidation have been connected with dif-ferent biological outcomes including convulsion in many other ani-mal species as well (Zhang et al., 2013). Saeed et al. (2010) studiedthe pattern of protein bands of Culex pipiens treated with Alliumsatvium, Citrus limon and Bacillus thuringiensis by comparing with

of Cedrus deodara oil on larval protein contents and its behavioral effect ons deodara oil –>. Saudi Journal of Biological Sciences (2017), http://dx.doi.

Fig. 6. Repellency test shows that bran mixed with Deodar extract deterredTenebrio molitor larvae.

I.D. Buneri et al. / Saudi Journal of Biological Sciences xxx (2017) xxx–xxx 5

Albino mice. According to them, 18 protein bands were found ininsect treated with 10ul of Allium satvium while in control batchthere were 13 bands after 12 h of treatment. Of these two bandswere common. After 24 and 48 h, the numbers of protein bandswere reduced to 17 and 15 respectively. According to our finding,8 protein bands appeared in the sample of Tenebrio molitor after48 h of treatment while control sample showed only 4 bands.Our study confirm the previous reports as it is cleared from boththat treated insects showed more protein bands and hence highprotein levels as compared to control. This may be due to defense,resistance mechanism or stressful condition due to which Tenebriomolitor larvae increase their protein levels.

Deodar oil in concentrated form can be used as insect repellentson infected livestock and an environment friendly bio-pesticide indifferent dilutions, as well. Deodar extract may take the place ofintramuscular injection used for tick’s infestation. Imidacloprid,Carbosulfan and deodar oil cause increase in protein levels andare also toxic against Tenebrio molitor larvae.

References

Bloomquist, J.R., 2009. Insecticides: chemistries and characteristics. In: Radcliffe, E.B., Hutchinson, W.D., Cancelado, R.E. (Eds.), Radcliffe’s IPM World Textbook.University of Minnesota, St. Paul, Minnesota.

Chaudhary, A., Sharma, P., Nadda, G., Tewary, D.K., Singh, B., 2011. Chemicalcomposition and larvicidal activities of the Himalayan cedar, Cedrus deodaraessential oil and its fractions against the diamondback moth, Plutella xylostella.J. Insect. Sci. 11, 1–10.

Please cite this article in press as: Buneri, I.D., et al. A comparative toxic effectlarvae of mealworm beetle (Tenebrio molitor) (Coleoptera: Tenebrionidae)Cedruorg/10.1016/j.sjbs.2017.06.005

Chapuis, M.P., Simpson, S.J., Blondin, L., Sword, G.A., 2011. Taxa-specific heat shockproteins are over-expressed with crowding in the Australian plague locust. J.Insect. Physiol. 57, 1562–1567.

Delbeke, F., Vercruysse, P., Tirry, L., De Clercq, P., Degheele, D., 1997. Toxicity ofdiflubenzuron, pyriproxyfen, imidacloprid and diafenthiuron to the predatorybug Orius laevigatus (Heteroptera: Anthocoridae). Biocontrol 42, 349–358.

Ezz, N.A., Fahmy, N.M., 2009. Biochemical effects of two kinds of mineral oils and anIGR on adult female Mealybug Ferrisia virgata (Cockerell). Egyptian Acad. J.Biolog. Sci. 1, 33–40.

Finney, D.J., 1971. Probit Analysis Second. Cambridge University Press, Kingdom, pp.68–78.

Gamble, J.S., 1922. A manual of Indian Timbers. Sampson Low. Marston and Co Ltd,London. UK.

Grainge, M.S., Ahmed, W.C., Mitchell Hylin, J.W., 1984. Plant Species ReportedlyPossessing Pest-control Properties. Á database. Resource Systems East-WestCenter.

Gregory, D.A., Johnson, D.L., Thompson, B.H., 1994. The toxicity of bran baits,formulated with carbaryl, chlorpyrifos and dimethoate, on yellow mealworms(Tenebrio molitor L.). J. Agri. Entomol. 11, 85–94.

Jacobson, M., 1982. Plants, insect and man—their interrelationship”. Econ. Bot. 36,346–354.

Kim, U., 2006. Neonicotinoid insecticides. MMG 445 Basic Biotechnol. J. 2, 46–52.King, A.M., MacRae, T.H., 2015. Insect heat shock proteins during stress and

diapause. Annual Rev. Entomol. 60, 59–75.Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the

head of bacteriophage T4. Nature, London 227, 680–685.Liu, Z., Williamson, M.S., Lansdell, S.J., Denholm, I., Han, Z., Millar, N.S., 2004. A

nicotinic acetylcholine receptor mutation conferring target-sit resistance toimidacloprid in Nilaparvata lugens (brown planthopper). Proceed. Nat. Acad. Sci.102, 8420–8425.

McCue, M.D., Guzman, R.M., Passement, C.A., Davidowitz, G., 2015. How and whendo insects rely on endogenous protein and lipid resources during lethal bouts ofstarvation? A new application for 13 C-breath testing. PloS One 10 (Pone0140053).

Makhaik, M., Naik, S.N., Tewary, D.K., 2005. Evaluation of anti-mosquito propertiesof essential oils. J. Sci. Indust. Res. 64, 129–133.

Marron, M.T., Markow, T.A., Kain, K.J., Gibbs, A.G., 2003. Effects of starvation anddesiccation on energy metabolism in desert and mesic Drosophila. J. Insect.Physiol. 49, 261–270.

Naqvi, S.N.H., Tabassum, R., Azmi, M.A., Hafeez, A., Tariq, R.M., Rashid, N., 1994.‘‘Histopathological effects of danitol (fenpropathrin) and neem fraction ofgrasshopper (Heteracris annulosa WlK) and changes in enzyme pattern. In:Proceed. 14th Cong. Zoo. (Khi). pp. 25–32.

Raguraman, S., Singh, D., 1997. Biopotentials of Azadirrachta indica and Cedrusdeodara oils on Callasobruchus chinensis. Int. J. Pharmacognosy. 35, 344–348.

Rahuman, A.A., Bagavan, A., Kamaraj, C., Vadivelu, M., Zahir, A.A., Elango, G.,Pandiyan, G., 2009. Evaluation of indigenous plant extracts against larvae ofCulex quinquefasciatus Say (Diptera: Culicidae). Parasitol. Res. 104, 637–643.

Morales-Ramos, J.A.M., Rojas, M.G., Shapiro-Ilan, D., Tedders, W.L., 2011. Self-selection of two diet components by Tenebrio molitor (Coleoptera:Tenebrionidae) larvae and its impact on fitness. Environ. Entomol. 40, 1285–1294.

Rao, I.G., Singh, D.K., 2002. Toxic effect of single and binary treatments of syntheticand plant-derived molluscicides against Achatina fulica. J. Appl. Toxicol. 22,211–215.

Saeed, R.M.A., Zayed, A.A., Namakay, A.H.E.H., Ismail, M., Mady, H.Y., 2010.Biochemical studies on Culex pipiens (L.) (Diaptera: Culicidae) exposed toAllium satvium, Citrus limon and Bacillus thuringiensis israelensis with referenceto assessement of the biosafety on Albino mice. Global Veterinaria 4, 22–23.

Singh, D., Rao, S.M., Tripathi, A.K., 1984. Cedar wood oil as a potential insecticidalagent against Mosquitoes. Naturwissenschaften 71, 265–266.

Singh, D., Rao, S.M., 1985. Toxicity of cedar wood oil against pulse beetle,Callosobruchus chinensis Linn. Indian Perfumer 29, 201–204.

Singh, D., Agarwal, S.K., 1988. Himachalol and himachalene: insecticidal principlesof Himalayan cedar wood oil. J. Chem. Ecol. 14, 1145–1151.

Singh, D., Siddiqui, M.S., Sharma, S., 1989. Reproduction retardant and fumigantproperties in essential oils against rice weevil (Coleopter Curculionidae) instored wheat”. J. Econ. Entomol. 82, 727–732.

Zhang, W., Xiao, S., Ahn, D.U., 2013. Protein oxidation: basic principles andimplications for meat quality. Crit. Rev. Food Sci. Nutr. 53, 1191–1201.

of Cedrus deodara oil on larval protein contents and its behavioral effect ons deodara oil –>. Saudi Journal of Biological Sciences (2017), http://dx.doi.